In the anterior pituitary lactotroph cells, transcription or PRL is regulated in part by a defined group of DNA-binding factors including Pit-1, Ets-1 and ERF. A family of corepressor proteins, including SMRT and N-CoR, interact with diverse classes of DNA-binding factors to silence target genes. Although N-CoR has been shown to repress the activity of Pit-1, the role of corepressors in the inhibition of PRL gene transcription has not been defined. The proposed studies will test the hypothesis that SMRT interacts with multiple DNA-binding factors to inhibit PRL transcription, and that dynamic interactions altering the subnuclear architecture are involved in this process. To begin identifying DNA-binding factors that mediate SMRT-dependent repression of the PRL gene, coimmunoprecipitation from GH3 cell lysates will be used to identify interactions between SMRT and either Pit-1, Ets-1 or ERF. Deletional analysis of the SMRT protein will be used to delineate functional domains. The effect of SMRT on the prolactin promoter and isolated response elements will be examined using reporter gene assays in GH3 cells and in HeLa cells transiently expressing the DNA-binding factors. Importantly, the dynamic sub-nuclear organization of SMRT and associated DNA-binding factors will be determined in living pituitary cells. This will be accomplished by the use of proteins tagged with color variants of the jellyfish green fluorescent protein. Additionally, SMRT subnuclear localization will be correlated with regions of transcriptional silencing. It is expected that these studies will have far-reaching implications for the regulation of PRL gene expression and for mechanisms of transcriptional repression in general.